Abstract

Glioblastoma multiforme (GBM) is an extremely malignant brain tumor. To identify new genomic alterations in GBM, genomic DNA of tumor tissue/explants from 55 individuals and 6 GBM cell lines were examined using single nucleotide polymorphism DNA microarray (SNP-Chip). Further gene expression analysis relied on an additional 56 GBM samples. SNP-Chip results were validated using several techniques, including quantitative PCR (Q-PCR), nucleotide sequencing, and a combination of Q-PCR and detection of microsatellite markers for loss of heterozygosity with normal copy number [acquired uniparental disomy (AUPD)]. Whole genomic DNA copy number in each GBM sample was profiled by SNP-Chip. Several signaling pathways were frequently abnormal. Either the p16(INK4A)/p15(INK4B)-CDK4/6-pRb or p14(ARF)-MDM2/4-p53 pathways were abnormal in 89% (49 of 55) of cases. Simultaneous abnormalities of both pathways occurred in 84% (46 of 55) samples. The phosphoinositide 3-kinase pathway was altered in 71% (39 of 55) GBMs either by deletion of PTEN or amplification of epidermal growth factor receptor and/or vascular endothelial growth factor receptor/platelet-derived growth factor receptor α. Deletion of chromosome 6q26-27 often occurred (16 of 55 samples). The minimum common deleted region included PARK2, PACRG, QKI, and PDE10A genes. Further reverse transcription Q-PCR studies showed that PARK2 expression was decreased in another collection of GBMs at a frequency of 61% (34 of 56) of samples. The 1p36.23 region was deleted in 35% (19 of 55) of samples. Notably, three samples had homozygous deletion encompassing this site. Also, a novel internal deletion of a putative tumor suppressor gene, LRP1B, was discovered causing an aberrant protein. AUPDs occurred in 58% (32 of 55) of the GBM samples and five of six GBM cell lines. A common AUPD was found at chromosome 17p13.3-12 (included p53 gene) in 13 of 61 samples and cell lines. Single-strand conformational polymorphism and nucleotide sequencing showed that 9 of 13 of these samples had homozygous p53 mutations, suggesting that mitotic recombination duplicated the abnormal p53 gene, probably providing a growth advantage to these cells. A significantly shortened survival time was found in patients with 13q14 (RB) deletion or 17p13.1 (p53) deletion/AUPD. Taken together, these results suggest that this technique is a rapid, robust, and inexpensive method to profile genome-wide abnormalities in GBM.(Mol Cancer Res 2009;7(5):665–77)

glioblastoma multiforme

genomic profiling

single nucleotide polymorphism

microarray

Introduction

Glioblastoma multiforme (GBM) is an extremely malignant subtype of astrocytoma, with survival times being <12 to 15 months. These tumors typically have a very high proliferative rate with widespread microvascular proliferation and areas of focal necrosis. Genetic abnormalities have been identified in GBM using cytogenetics, fluorescence in situ hybridization, and comparative genomic hybridization. These studies have shown several notable abnormalities. The p16(INK4A)/p15(INK4B)-CDK4/6-pRb pathway was found to be aberrant in the vast majority of GBMs either as a result of inactivation of either p16(INK4A) or Rb or overexpression of either CDK4 or CDK6 (1, 2). Homozygous deletion of p16(INK4A) occurs in approximately 31% to 50% of GBMs (3, 4). The CDK4 gene is amplified on chromosome 12q13-14 in ∼15% of GBM (1). The long arm of chromosome 13 is lost in nearly 33% to 50% of these tumors; the Rb gene is at least one of the targets in this deletion, being inactivated in ∼35% of samples (5-8). Loss of chromosomal material on chromosome 10 occurs in 60% to 95% of GBMs (9). One common minimum deleted region (CMDR) at 10q23.3 includes the PTEN gene, which occurs in approximately 20% to 30% of samples (10-12). Epidermal growth factor receptor (EGFR) is amplified in ∼40% of GBMs (9). Of interest, 33% of these tumors with EGFR amplification have a specific EGFR rearrangement, producing a smaller protein, making it similar to the v-erbB oncogene (13). Amplification of EGFR is associated with overexpression of the EGFR protein and it is often associated with deletion of PTEN and p16(INK4A) (14). Alterations of chromosome 17p are associated with a mutation of the p53 gene in ∼30% of GBMs. The prominent vascularity that occurs in GBM is probably the result of excess expression of growth factors and their receptors [e.g., vascular growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR)] that are associated with angiogenesis.

We used high-density (50K/250K) arrayed oligonucleotide probes that contain single nucleotide polymorphisms (SNP), the so-called SNP-Chip, to identify genetic abnormalities, including amplicons, duplications, deletions, and acquired uniparental disomy [AUPD; loss of heterozygosity (LOH) with normal copy number]. Our SNP-Chip results were validated using several other techniques, including quantitative PCR (Q-PCR), nucleotide sequencing, and detection of LOH by microsatellite markers. Using SNP-Chips, we identified aberrant genes as well as aberrant genetic pathways in GBM. This robust technology should set the stage to formulate new diagnostic subcategorizations of GBM and to provide prognostic indicators for physicians to set up personalized therapy according to the aberrant genetic pattern of the GBM of the individual.

Results

Signaling Pathway Abnormalities Present in GBM

DNA from 55 GBMs (22 with matched normal peripheral blood neutrophils) and 6 GBM cell lines were examined by SNP-Chip assay for genomic abnormalities (Tables 1 and 2). Common duplications and deletions of either segments or entire chromosomes were observed. For example, chromosome 7 was duplicated in 30 of 55 GBM samples, and deletion of either all or part of chromosome 10 occurred in 33 of 55 GBM samples. Also, small common minimally deleted regions (CMDR) or small common minimally amplified regions (CMAR) that involved only one or several genes were found. For example, 36 of 55 clinical samples had a deletion at chromosome 9p21.3 containing the p14(ARF), p15(INK4B), and p16(INK4A) tumor suppressor genes (Fig. 1A); notably, 20 of these 36 GBM samples had homozygous deletions at this site. Also, 36 of 55 samples had either duplication (19 samples) or amplification (17 samples) at chromosome 7p11.2, which contained the EGFR gene (Fig. 1C; five representative samples). The CMDRs and CMARs were confirmed by reverse transcription Q-PCR (Fig. 1B and D). All the CMDRs and CMARs are summarized on Table 1. For 51 of the 55 GBM patients, survival data were available. Median survival time was 60 weeks. Overall, 39 events were reported. Significant short survival time (P values were not corrected for multiple testing) was found in the patients with 13q14 (RB) deletion (Fig. 1E) or 17p13.1 (p53) deletion/AUPD (Fig. 1F). In these CMDR and CMAR, the genes involve in the p16(INK4A)/p15(INK4B)-CDK4/6-pRb (pathway 1) and the p14(ARF)-MDM2/4-p53 (pathway 2) were frequently aberrant: pathway 1 had deletion of p16(INK4A) and p15(INK4B) in 66% of cases; deletion of Rb in 40% of cases; and amplification of CDK4 in 11% and trisomy of CDK6 in 55%. Pathway 2 had deletion of p14(ARF) in 66% cases, deletion or AUPD of p53 in 33%, and amplification of MDM2 in 11% and trisomy or amplification of MDM4 in 13%. Alteration of MDM2 and MDM4 was mutually exclusive in the same sample. Taken together, alterations of p16(INK4A)/p15(INK4B)-CDK4/6-pRb pathway occurred in 48 of 55 (87%) cases, and the p14(ARF)-MDM2/4-p53 pathway was abnormal in 48 of 55 (87%) samples; both pathways were simultaneously aberrant in 46 of 55 (84%) cases. In addition, the pathway associated with tyrosine kinase receptor signaling (pathway 3), including deletion of PTEN or amplification of EGFR and/or VEGFR/PDGFR α (PDGFRA), was detected in 39 of 55 GBM samples (71%).

Representative SNP-Chip analysis and validation of GBM samples. A. 9p21.3 deletions: Four representative patterns of chromosome 9p21.3 deletions in GBM samples detected by SNP-Chip. Blue lines above each chromosome show total gene dosage; level 2 indicates diploid (2N) amount of DNA, which is normal. Green bars under each chromosome indicate the SNP sites showing heterozygosity in GBM samples. When heterozygosity is not detected in the tumor but is found in its matched normal control, the result suggests that the GBM has allelic imbalance in that region. The bottom lines in each panel show allele-specific gene dosage (one line indicates gene dosage of the paternal allele and the other indicates the gene dosage of the maternal allele). Level 1 is normal for each gene dosage. Six representative samples with 9p21.3 homozygous deletion with a CMDR containing CDKN2A and CDKN2B are shown. B. Data validation: DNA dose of CDKN2A was measured by Q-PCR. Stripe columns, the DNA dose in GBM samples; black columns, DNA dose in their matched normal controls. Each sample showed homozygous CDKN2A deletion by SNP-Chip analysis. C. Chromosome 7p11.2 amplicon: Four representative GBM samples with amplification at chromosome 7p11.2 are shown. The CMAR contains EGFR, ECOP, LANCL2, FKBP9L, GBAS, PSPH, CCT6A, SUMF2, PHKG1, and CHCHD2 genes. D. Data validation: DNA dose of EGFR was measured by Q-PCR. Stripe columns, relative DNA amounts in GBM samples; black columns, DNA dose in their matched normal controls. Each sample showed EGFR amplification by SNP-Chip analysis. Kaplan-Meier estimates of overall survival for patients with and without 13q14 Del (RB; E) and 17p13.1 Del/AUPD (p53; F). Tick marks indicate censored data. Prognosis is worse for patients with a deletion of 13q14 (P = 0.0224 by the log-rank test) and deletion/AUPD of 17p13.1 (P = 0.00603).

SNP-Chip array of chromosome 2 from the U118 GBM cell line showed a deletion in the LRP1B gene, spanning exons 3 to 18 (Fig. 2). Nucleotide sequencing of the cDNA of LRP1B showed a truncated LRP1B in which exons 2 and 19 were fused and an early stop codon occurred, resulting in only a 75-amino-acid open reading frame. Internal deletion of LRP1B also occurred in four GBM samples (representative samples shown in Fig. 2). The results suggest that internal deletion of LRP1B is associated with development of glioma. We also found that one sample had marked amplification of chromosome 5p13.2 (Fig. 3A). About 20 genes are localized in this amplicon, including IL7R, CAPSL, SKP2, and SLC1A3. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27(KIP1). We measured mRNA levels of SKP2 in 21 GBM tumor samples and 6 normal brain samples; the gene was highly expressed in 52% (11 of 21) of the GBM samples (>2-fold the average level of six normal brain samples; Fig. 3B).

Internal deletion of LRP1B in GBM. Representative deletions of the LRP1B gene are shown in the U118 GBM cell line and two GBM samples. The cDNA of LRP1B from U118 was sequenced, showing a deletion between exons 3 and 18. This truncated cDNA has an early stop codon after amino acid 105 (TAA). The sequence that is underlined is at exon 19.

Deletion at 1p36.23 and 6q26-27 in GBM

A total of 19 of 55 GBM samples had a deletion that included 1p36.23; 11 had a small heterozygous and 3 had a homozygous deletion at this region involving two CMDRs (Fig. 4). Genes within these CMDRs include CAMTA1, PER3, UTS2, TNFSF9, VAMP3, PARK7, MIG6, RERE in one region, and GPR157, H6PD in the second region (Fig. 4). One GBM (case c19) had a homozygous deletion in the intron between exons 3 and 4 of CAMTA1.

Another common deleted region occurred in 6q. Loss of 6q was discovered in 16 of 55 samples. Two of these GBMs had a homozygous deletion at 6q26-27, including the PARK2, PACRG, QKI, and PDE10A genes (Fig. 5A). The expression of PARK2 in 34 of 56 additional GBM samples (61%) was lower than the 50% mean level present in normal brain tissue (Fig. 5B).

AUPD at 17p in GBM

One of the advantages of the SNP-Chip technique is the ability to identify AUPD. During carcinogenesis, the abnormal allele is often duplicated and the normal matched allele is deleted, resulting in the duplication of the abnormal allele. A total of 32 AUPDs were found in 55 tumor samples and eight of these AUPDs were at the same region of 17p, which included the p53 gene (Fig. 6A). The 17p AUPDs were validated using several different approaches. As might be predicted, the quantity of DNA at this 17p chromosomal region was normal when comparing the samples having AUPD with their matched normal DNAs (Fig. 6B). Moreover, LOH analysis using a microsatellite marker on 17p13.3 showed loss of one of the normal alleles when comparing tumor and matched normal control DNA (Fig. 6C). Homozygous p53 mutations (from exons 5 to 8) were identified by nuclear sequencing the shifted bands identified by single-strand conformational polymorphism (SSCP). Of the eight tumor samples with AUPD, one sample (S1) had a 254I deletion, another (S9) had a R175H mutation (Fig. 6D), and two cases had a R248Q mutation. Also, 17p AUPD encompassing the p53 gene was found in the U118, U138, U343, U373, and T98G GBM cell lines. Nucleotide sequencing showed that each of these cell lines had a homozygous mutant p53 with loss of the normal p53 allele (Fig. 6D and data not shown). In contrast, no chromosome 17p AUPD was found in the U87 GBM cell line, and this cell line had a wild-type p53 gene (data not shown).

Chromosome 17p13.3-12 AUPD in GBM. A. Two representatives examples of AUPD at chromosome 17p13.3-12. B. DNA level of p53 was measured by real-time quantitative PCR. Striped and black bars, DNA amount in GBM samples and their matched normal control, respectively. C. Microsatellite marker (chr17:1857382-1857419) was used to analyze LOH in tumor samples compared with their normal matched controls (, band pattern in normal samples; , band pattern in tumor samples). D. DNA of three GBM samples was analyzed for p53 mutations initially by SSCP (, band pattern in normal samples; , band pattern in tumor samples). Nucleotide sequencing: G changed to A in S9; deletion of CAT in S1; and G changed to A (M237R) in T98G (sequence data not shown).

Discussion

High-density SNP-Chip arrays allow rapid detection of amplifications, deletions, and AUPD. This novel technique greatly increases sensitivity of detection of copy number changes in small regions of the genome using only 250 ng genomic DNA. A 250K SNP-Chip interrogates on average about every 12-kb genome, often allowing detection of changes within a gene. Comparative genomic hybridization (CGH) is another technique to examine tumors for an overview of the genome. CGH has been used to detect the chromosome imbalances and molecular classification in gliomas (15-17). Unlike CGH, SNP-Chip technique can detect AUPD. As shown by our study, AUPD is very frequent in GBM. Two previous studies also analyzed genomic abnormalities in gliomas by the SNP-Chip technique (4, 18). One of these was a multi-institutional study that incorporated several genomic approaches, including SNP-Chip, sequencing, and microarray expression analysis (4). Their studies and ours confirmed that this technique is a useful tool to identify novel genomic changes in GBM and a number of common abnormalities were identified including regions of AUPD.

AUPD can be identified by LOH analysis plus quantification of the DNA within the region; however, these abnormalities are more quickly and robustly identified by SNP-Chip analysis. A study of various cancer cell lines found on average 4.7 AUPDs per cell line (19). AUPD results in a duplicated chromosomal region containing either a mutated, inactivated tumor suppressor gene or mutated, activated oncogene. For example, we found frequent AUPD at 17p in GBM samples that contained a mutant p53; thus, the AUPD resulted in duplication of the mutant p53 and loss of the normal p53 allele. GBMs seem to favor duplicating the mutant p53 (AUPD) rather than merely deleting the normal allele and keeping one copy of the mutant p53. Missense mutation resulting in an expressed protein with a long half-life is the most frequent alteration in cancer (20). The p53 protein forms tetramers. Mutant p53 might either behave in a dominant-negative fashion or have a gain of function and contribute to oncogenic activities in vitro and in vivo (21). For example, knock-in mice carrying one null allele and one mutant allele of the p53 gene developed novel tumors compared with p53-null mice (22-26).

A recent SNP-Chip study showed that the EGFR located at chromosome 7p11.2 was amplified in up to 43% GBM samples (4), and amplification of the gene was associated with overexpression of the EGFR protein in GBM (9). We found that 19 of 55 GBMs (35%) had trisomy of chromosome 7 or duplication of chromosome 7p, and another 17 of 55 (31%) GBM had prominent amplification of the EGFR gene. The SNP-Chip analysis showed that at least nine additional genes were coamplified with EGFR. Of note, some breast cancers with amplification of Her2/neu (EGFR-2) also contain the topoisomerase 2 (TOPO-2) gene in the same amplicon. These patients have an increased response when treated with both an inhibitor of TOPO-2 and trastuzumab (Herceptin, a monoclonal antibody that targets Her-2/neu; ref. 27). Further studies of the genes in the EGFR amplicon may offer additional therapeutic targets for GBM. Also, VEGFR and PDGFRA were in an amplicon or involved in trisomy on chromosome 4q11-12 in 5 of 55 (9%) GBM samples. In a recent study, high-level amplification of VEGFR/PDGFRA occurred in 13% samples (4). Amplification and presumably abundant expression of these genes may allow for autocrine and paracrine stimulation of growth. These tumors may be more sensitive to inhibitors of these receptors or their growth factors [e.g., bevacizumab (Avastin)].

We found that the SKP2 gene on chromosome 5p was markedly amplified in a GBM sample and its expression was increased >2-fold compared with normal brain tissue in 11 of 21 additional GBM samples. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27 (28). Saigusa et al. reported amplification of DNA at chromosome 5p in the region of SKP2 in four glioma cell lines and also found that the expression of the SKP2 gene was increased in 31% of primary GBM examined by immunohistochemistry (29). They found that increased expression of SKP2 was associated with an overall shorter survival (29). High levels of SKP2 have also been associated with cancers of the thyroid (30) and the lung (31, 32), as well as neuroblastomas (33).

The short arm of chromosome 1 at 1p36 is frequently deleted in hematopoietic malignancies (34, 35), epithelial cancers (36, 37), and neural-related cancers, including neuroblastomas (38), meningiomas (39), and gliomas (40). Allelic loss at 1p has been reported in 20% to 30% of astrocytomas. Other investigators defined a 150-kb CMDR on 1p36.23 by LOH mapping, which encompassed CAMTA1, a gene encoding a transcription factor (41). We also discovered 1p deletions in 19 of 55 GBM samples (35%), which included two CMDRs. One contained the CAMTA gene alone or the CAMTA gene plus PER3, UTS2, TNFSF9, VAMP3, PARK7, MIG6, and RERE; the second CMDR had both of the GPR157 and H6PD genes in a sample. Further studies are required to determine if CMTA is the key target gene of 1p36 deletion in various cancers including GBM.

The LRP1B gene is highly homologous to the lipoprotein receptor-related protein 1 (LRP1, a family member of the low-density lipoprotein receptors). LRP1B has been classified as a tumor suppressor gene and is frequently mutated in GBM (42-47). We discovered a novel internal deletion of LRP1B in the U118 GBM cell line and four GBM samples. Nucleotide sequencing of the LRP1B gene from U118 cells showed loss of exons 3 to 18 and an early stop codon, suggesting that the protein was no longer functional. Other studies have suggested that LRP1B may be a tumor suppressor gene that is deleted or abnormal in several other tumor types, as well as GBM (42-47). In addition, LRP1B is deleted or epigenetically silenced in oral urothelial, esophageal, and non–small-cell lung cancers; taken together, LRP1B behaves as a tumor suppressor gene that is frequently mutated in several solid tumors, including GBM (42-47). Our data suggest that LRP1B acts as a tumor suppressor gene in glioma cells and is aberrant in GBM.

Deletion of ∼165 Mb of chromosome 6q25-27 in GBM involving IGF2R, PARK2, PACRG, and QKI was found by CGH (40). Our SNP-Chip data showed that 16 of 55 (29%) GBM samples had deletion of chromosome 6q and two of these samples had a homozygous deletion (4%) at 6q26-27, which included the PARK2, PACRG, QKI, and PDE10A genes. In another large study, 54 deletions of this region were found in a total of 206 (26%) GBM samples, including six homozygous deletions (3%; ref. 4). The PARK2 gene has been reported to be mutated in some patients with autosomal recessive juvenile Parkinson's disease, and investigators recently suggested that the gene is a candidate tumor suppressor gene in ovarian and breast cancers as well as leukemias (48-50). PARK2 shares a bidirectional promoter with PACRG. Abnormal methylation of this common promoter was observed in 26% of acute lymphoblastic leukemia and 20% of chronic myelogenous leukemia in lymphoid blast crisis and was associated with decreased expression of both of these genes (48). We found that ∼61% (34 of 56) of GBM samples had 50% decreased expression of PARK2 compared with normal control brain tissues. Our data suggest that PARK2 behaves as a tumor suppressor gene that is inactivated in GBM.

In a large comprehensive study, deletion at chromosome 10q23.3 (contains PTEN gene) was found in ∼173 of 206 (84%) GBMs, including homozygous deletions of the PTEN gene in 9 of 206 (4%) samples (4). Further studies found that PTEN was mutated in 44% of GBM, and 60% of those with LOH at 10q had a mutant PTEN gene (51). PTEN is an inhibitor of the phosphoinositide 3-kinase mammalian target of rapamycin/AKT pathway that is downstream of EGFR or VEGFR/PDGFRA in this growth stimulatory pathway. In our study, 33 of 55 (60%) GBM samples had lost a PTEN allele. Two samples (4%) had homozygous deletion of this gene. Taken together, 71% of GBM samples had either deletion of PTEN or amplification/trisomy of EGFR and/or VEGFR/PDGFRA.

The cyclin-dependent kinase inhibitor p16(INK4A) normally inhibits CDK4 and CDK6, resulting in dephosphorylation of Rb. This ensures that Rb continues to bind and inactivate E2F (cell cycle transcription factor), maintaining a brake on the cell cycle. The p16(INK4A)-CDK4/6-Rb pathway is aberrant in the vast majority of GBMs (1, 2). The p16(INK4A), p15(INK4B), and p14(ARF) (inhibitor of MDM2) genes are contiguous on chromosome 9. We found that all three genes were lost in 35 of 55 (64%) GBM samples, either by homozygous or hemizygous deletion. Also, 30 of 55 GBMs had trisomy of chromosome 7, resulting in three copies of CDK6; 6 of 55 GBM samples had amplification of CDK4 on chromosome 12. In addition, the Rb gene (chromosome 13q14.2-14.3) was deleted in 22 of 55 GBM samples. Taken together, the p16(INK4A)/p15(INK4B)-CDK4/6-pRb pathway was structurally aberrant in 48 of 55 GBM (87%) samples.

The p14(ARF) binds to MDM2 and MDM4, preventing them from decreasing the level of p53. Loss of p14(ARF) or amplification of MDM2/MDM4 lead to inactivation of p53. We found that either the MDM4 (1q32.1) or the MDM2 (12q15) gene was increased in copy number in 13 of 55 (24%) GBMs, p14(ARF) was deleted in 35 of 55 (64%) cases, and 18 of 55 (33%) samples had either AUPD or deletion of p53. Thus, the p14(ARF)-MDM2/4-p53 pathway was structurally aberrant in 48 of 55 GBM (87%) samples. Ghimenti et al. noted alterations of the p14(ARF)-MDM2-p53 pathway in 73% of GBM samples (52). We found that the frequency of amplification/trisomy of MDM4 was similar to amplification of MDM2 in GBM samples and both rarely coexisted in the same sample. This is consistent with previous reports (53, 54). Taken together, abnormalities of the EGFR/VEGFR/PDGFRA-PTEN (71%), p16(INK4A)/p15(INK4B)-CDK4/6-pRb (87%), and p14(ARF)-MDM2/4-p53 (87%) pathways were present in the vast majority of GBM samples, and almost all GBM (91%) samples had an abnormality of at least one of the three pathways (Fig. 7). Congruent with our study, alteration in these three core pathways was noted in 78% to 88% of GBM samples in a large cooperative study (4). The proteins of these pathways provide therapeutic targets for GBM. These include EGFR inhibitors, such as erlotinib and gefitinib (55), dual inhibitors of PI3K and mTOR, including the investigational drug PI-103 (56) and inhibitors of mTOR [e.g., rapamycin (57) and Everolimus (58)].

The aberrant genes in GBM are incompletely identified. Present-day clinical and therapeutic management often relies on 50- to 100-year-old techniques of morphology and immunochemistry. SNP-Chip is an extremely robust technology, offering the opportunity to discover, in a potentially simple fashion, copy number changes, including AUPD associated with GBM. The patterns of genetic abnormalities, even in the absence of knowledge of the specific genetic lesions, may result in a useful, new GBM classification. Furthermore, new genetic lesions identified by SNP chip may become targets for novel therapies.

Materials and Methods

Clinical Samples, Cell Cultures, and DNA Preparation

GBM from 55 individuals were studied by SNP-Chip. The age of these patients ranged from 18 to 70 years. Twenty-two of these 55 samples had matched peripheral blood neutrophils from the same patients. Also, 4 of the 55 GBM samples were established explants as previously described (59) and used for SNP analysis. Furthermore, six human GBM cell lines (U87, U118, U138, U343, U373, and T98G) were used for SNP-Chip studies. Standard proteinase K-phenol-chloroform extraction method was used to extract DNA from GBM samples, cell lines, and explants. In addition, 56 additional GBM samples were quick frozen, and RNA was extracted and used for expression analysis of target genes. Written informed consent for research use of all of these samples was obtained before surgery, according to a protocol approved by the institutional ethics committee. Cell lines were maintained in DMEM (Life Technologies) with 10% FCS (Gemini Bio-Products), 10 units/mL penicillin G, and 10 mg/mL streptomycin (Gemini Bio-Products). All cells were incubated at 37°C in 5% CO2.

High-Density SNP-Chip Analysis

SNP-Chips for human 50K XbaI/250K Nsp arrays were used for this study (SNP-Chip, Affymetrix). The DNA samples from 22 GBM patients with matched peripheral blood neutrophils from the same patient as control DNA were analyzed by 50K SNP-Chips as previously described (60). The other 33 DNA samples without matched control DNA and six human GBM cell lines were analyzed by 250K SNP-Chips, using allele-specific copy number analysis using anonymous references (AsCNAR; ref. 61). Fragmentation and labeling of DNAs were done using a GeneChip resequencing kit (Affymetrix); hybridization, washing, and signal detection were done on a GeneChip Fluidics Station 400 and GeneChip scanner 3000 according to the manufacturer's protocols (Affymetrix). Microarray SNP data were analyzed for determination of both total and allelic-specific copy number using the CNAG program with AsCNAR capability,7 as we have previously described (47, 48). In the latter algorithm, AsCNs were estimated using “nonpaired” genomic DNA as controls, where array signals at the heterozygous SNP sites in the tumor sample are compared with the corresponding signals in one or more control samples also showing the heterozygous SNP calls. All the SNPs within an inferred LOH region are formally analyzed as heterozygous SNPs (see ref. 48 for mathematical details). Control sample sets used for allelic-specific copy number calculations may be different from SNP to SNP. AsCNAR enables detection of LOH regions in those samples highly contaminated with normal cells and provides an accurate measurement of total copy numbers (48). Size, position, and location of genes were identified with the University of California, Santa Cruz Genome Browser.8 Our software allows accurate calls of AUPD and copy number changes without requiring germline DNA. All known copy number polymorphism were eliminated from analysis by using the UCSC Genome Browser. All the raw data of these SNP-Chips have been submitted to the public database ArrayExpress.9 The ArrayExpress accession number is E-MEXP-1330.

Quantitative-PCR

Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's protocol. DNA was removed by DNase. Two micrograms of RNA were reverse transcribed with random primers and Superscript III reverse transcriptase (Invitrogen). The cDNA was used for real-time PCR with Platinum Taq (Invitrogen) and SYBRGreen I (Molecular Probes) in triplicates in an iCycler iQ system (Bio-Rad). The PCR conditions were as follows: 2 min at 94°C followed by 45 cycles of 94°C for 15 s, 60°C for 15 s, 72°C for 15 s, and fluorescence determination at the melting temperature of the product for 20 s. Specificity of PCR products was checked on agarose gel. The expression of β-actin was used as an endogenous reference. A comparative threshold cycle was used to determine target genes and β-actin gene expression relative to the no-sample control (calibrator). The mRNA expression level was normalized by β-actin expression. The relative expression level of target genes in control samples was normalized to a relative value of 1. Final results in treated samples were expressed as n-fold difference to the control samples. The DNA dose also was measured by real-time quantity PCR as described above. The sequences of primers are shown in Table 3.

Oligonucleotide Primer Sequences Used for Real-time Q-PCR for DNA and cDNA

SSCP and Nucleotide Sequencing

For detection of p53 mutations using SSCP, each PCR reaction contained 20 ng DNA, 10 pmol of each of the primers, 2 nmol/L of each of deoxynucleotide triphosphate, 0.5 units of Taq DNA polymerase, and 3 μCi [α-32P]dCTP in 20 μL. PCR products were diluted 10-fold in the loading buffer containing 10 mmol/L NaOH, 95% formamide, and 0.05% of both bromophenol blue and xylene cyanol. After denaturation at 94°C for 5 min, 2 μL of the samples were loaded onto a 6% nondenaturation polyacrylamide mutation detection enhancement gel (MDE Bioproducts) with 10% (v/v) glycerol and electrophoresed at 4 to 6 W for 20 h. Subsequently, the gel was dried and subjected to autoradiography. Single-strand DNAs of mutant candidate bands showed aberrant migration through SSCP analysis. These shifted bands were excised and eluted by diffusion in 50 μL TE buffer. One microliter of elution TE was used as template to reamplified the fragment. The resulting product was sequenced using the Big-dye sequence reaction (Applied Biosystems) and analyzed on an Autosequencer 3100. The p53 gene was analyzed for mutations in exons 5 to 8 (62).

LOH Analysis

PCR amplification of microsatellite sequences was used to determine LOH on chromosome 17p. Primers for amplification of the microsatellite fragments (chr17:1857382-1857419) were 5′-CGCCTTTCCTCATACTCCAG and 5′-GCCAGACGGGACTTGAATTA. Each PCR reaction contained 25 ng of DNA, 10 pmol of each primer, 2 nmol of each deoxynucleotide triphosphate, 0.5 units of Taq DNA polymerase, and 3 μCi of [α-32P] dCTP in 20 μL of the specified buffer with 1.5 mmol/L MgCl2. Thirty-two cycles of amplification, PAGE, and subsequent autoradiography were done as published previously (63).

Statistical Analysis

Overall survival was analyzed with the Kaplan-Meier method to assess prognostic significance of the observed genomic abnormalities. We applied the log-rank test to compare patients with and without a specific genomic abnormality. P values were not adjusted for multiple testing. Analyses were done in R10 using survival package version 2.34-1.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

. Abnormal methylation of the common PARK2 and PACRG promoter is associated with downregulation of gene expression in acute lymphoblastic leukemia and chronic myeloid leukemia. Int J Cancer2006;118:1945–53.